To complement the currently available cold behavioral assays, we have developed a novel high- throughput behavioral assay to measure the responses of unrestrained mice to cold stimuli. Mice are acclimated in plastic enclosures on a borosilicate glass plate, and the glass directly underneath the rear hind paw is rapidly cooled with a dry ice pellet until the mouse withdraws its paw. The latency to withdrawal from the dry ice stimulus is quantified as a measure of the temperature at which the mouse responds to cold. This assay will be used to provide unambiguous information about the cold sensitivity of TrpM8-/-, TrpA1-/-, and TrpM8-TrpA1 double knockout mice, and therefore about the roles of TrpM8 and TrpA1 in cold sensation. To better understand which neuronal populations are important for the transmission of these signals, we use optogenetic techniques to transiently silence specific, discrete populations of sensory neurons in the periphery without any surgical intervention or altering the neuronal architecture. Using this technique, we silence specific neuronal populations while applying the cold stimulus from our new assay, allowing us to test the role of each of those populations in the response to the cold stimulus. PUBLIC HEALTH RELEVANCE: Cold sensation and pain are clinically important to chemotherapy, neuropathy, and stroke patients among others. Improved understanding of the neuronal populations that are involved in the transduction of cold stimuli, as well as molecular mechanisms they employ, will allow the development of treatments with better efficacy and fewer off-target effects.